Ectodomain (ECD) cleavage by metalloproteinases is involved in many diseases including kidney and cardiac disease, Alzheimer's disease, cancer and inflammation. EGF pro-ligands mature by metalloproteinase-cleavage of the ECD that represents the active ligand. They are important in a broad range of physiological and disease states. In the kidney, as examples, the EGF ligand TGFalpha is cleaved in response to Angiotensin II (Angll). TGFalpha knock-out mice or mice treated with a metalloprotease inhibitor are protected against Angll-induced chronic kidney disease. HB-EGF, another EGF ligand, is involved in tubular branching in the developing kidney and in tubular repair after kidney injury. Metalloproteinase-directed therapies in humans are limited by side effects, or non-existent. The signal transduction pathways regulating ECD cleavage are essentially unknown. The goal of our study is to identify novel genes that regulate ECD cleavage using a lentiviral shRNA gene knock-down approach. We have developed a high-throughput assay that detects cleavage of EGF-ligands in a FACS-based assay using cells stably expressing a ligand with an ECD epitope tag and a C-terminal GFP-fusion. The ECD can be tracked by staining with a fluorochrome-coupled antibody (red). In the uncleaved state any given cell has a 1:1 ratio of outside (ECD, red) to inside (GFP, green) fluorescence, as measured by FACS on life single cells. Stimulation of EGF ligand cleavage decreases, while inhibition increases this ratio. During the K99 phase of this grant we began examining the effect of knock-down of human kinases and phosphatases on EGF ligand cleavage. Here we present up-to-date results on the initial screen that identified several inhibitors and activators of phorbol ester-induced TGFalpha cleavage in Jurkat cells. Knock-down of PKCzeta, an atypical protein kinase C isoform not activated by phorbol ester is identified as an important inhibitor of cleavage in Jurkat cells and also in mouse lung epithelial cells and partipates in differential regulation of ECD cleavage of three different EGF ligands examined. We are outlining our plans for completion of the high-throughput screen and for further evaluation of candidate genes, including PKCzeta.